Towards single cell ICP-MS normalized quantitative experiments using certified selenized yeast.

In the search for a normalized procedure to replicate and compare single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) experiments, SELM-1, a certified reference material containing selenium enriched yeast cells has been used. Selenium concentrations (both, intra- and extracellular) have been measured using either sequential or simultaneous procedures. Regarding quantitative results, the sequential procedure involving cell washing followed by freeze drying of the washed material and intracellular Se quantification using SC-ICP-MS provided best results. In this case, intracellular Se accounted for 1304 ± 48 mg kg-1 (corresponding to 64% of the certified Se content). The average mass of Se per yeast cell was 41.6 fg Se with a dispersion of 1.6-279 fg Se/cell. In the isolated extracellular Se fraction, the Se concentration accounted for 412 ± 48 mg kg-1 (about 21% of the total Se). Thus, the sequential procedure provided a total Se recovery of about 85% with respect to the certified value. The direct dilution and simultaneous measurement of intra- and extracellular Se by SC-ICP-MS provided results of 1024 ± 42 mg kg-1 for intracellular and 316 ± 30 mg kg-1 for extracellular Se representing a total recovery of about 66%. In both cases, an initial thorough characterization of the cell density per solid weighed material was conducted by flow cytometry and the cell integrity ensured using confocal microscopy. These results clearly demonstrated that with appropriate sample preparation, SC-ICP-MS is a unique tool, which is capable of providing quantitative information about intracellular and extracellular Se. In addition, SELM-1 seems the ideal tool to enable data normalization at the single cell level to replicate, benchmark, and improve new SC-ICP-MS studies by using the same material for data validation.

Evaluation of copper uptake in individual spores of Streptomyces coelicolor and endogenic nanoparticles formation to modulate the secondary metabolism.

Copper modulates secondary metabolism in Streptomyces. Although the cytosolic copper concentration is controlled by several chaperones and transporters, the formation of copper nanoparticles (NPs) and its relation to the antibiotic production has never been established in the model Streptomyces coelicolor. In this work, state-of-the-art analytical tools are used to evaluate the incorporation of copper in individual spores of S. coelicolor at different exposure concentrations (40, 80, and 160 µM Cu). Among them, the use of single cell-inductively coupled plasma-mass spectrometry revealed incorporation levels in the range of 2 to 2.5 fg/spore (median) increasing up to 4.75 fg/spore at the upper exposure concentrations. The copper storage within the spores in the form of NPs was evaluated using a combination of single particle-inductively coupled plasma-mass spectrometry and transmission electron microscopy. The obtained data confirmed the presence of NPs in the range of 8 to 40 (mean size 21 nm) inside S. coelicolor spores. The presence of the NPs was correlated with the actinorhodin production in liquid non-sporulating cultures amended with up to 80 µM Cu. However, further increase to 160 µM Cu, yielded to a significant decrease in antibiotic production. Secondary metabolism is activated under stressful conditions and cytosolic copper seems to be one of the signals triggering antibiotic production. Particularly, NP formation might contribute to modulate the secondary metabolism and prevent for copper toxicity. This work describes, for first time, the formation of endogenous copper NPs in S. coelicolor and reveals their correlation with the secondary metabolism.

Targeting HER2 protein in individual cells using ICP-MS detection and its potential as prognostic and predictive breast cancer biomarker.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane protein that has become one of the most specific prognostic and predictive biomarker of breast cancer. Its early detection is key for optimizing the patient clinical outcome. This work is focused on the detection of HER2 in individual cells using an antibody containing lutetium (Lu) as reporter group that is monitored by introducing the individual cells into the inductively coupled plasma mass spectrometer (ICP-MS). This Lu-containing antibody probe is used to label different breast cancer cell lines considered HER2 negative (MDA-MB-231) and positive (SKBR-3 and BT-474). Optimizations regarding the amount of the probe necessary to ensure complete labelling reactions are conducted in the different cell models. Concentrations in the range of 0.006 fg Lu/cell and 0.030 fg Lu/cell could be found in the HER2 negative and HER2 positive cells, respectively. In addition, the selectivity of the labelling reaction is tested by using two different metal-containing antibody probes for HER2 (containing Lu) and for transferrin receptor 1 (containing Nd), respectively, within the same cell population. Finally, the methodology is applied to the targeting of HER2 positive cells in complex cell mixtures containing variable amounts of BT-474 and MDA-MB-231 cells. The obtained results showed the excellent capabilities of the proposed strategy to discriminate among cell populations. This finding could help for scoring HER2 positive tumors improving existing technologies.

Ultrasmall iron oxide nanoparticles cisplatin (IV) prodrug nanoconjugate: ICP-MS based strategies to evaluate the formation and drug delivery capabilities in single cells.

Ultrasmall iron oxide nanoparticles (<10 nm) were explored here as nanotransporters of cis-diamminetetrachloroplatinum (IV) (a cisplatin prodrug) in cellular models. The coating of the particles containing reactive carboxylic acid groups enabled the formation of a stable conjugate between the prodrug and the nanoparticles using one pot reaction. The nanoconjugate was characterized by different techniques exhibiting diameters of about 6.6 ± 1.0 nm. The use of a hyphenated strategy based on high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) permitted the quantitative evaluation of Fe and Pt in the nanoconjugate. Furthermore, the cellular uptake of the synthetic nanoconjugate was explored by single cell-ICP-MS (SC-ICP) which was used for the first time in this type of studies. The experiments in A2780 and A2780cis, sensitive and resistant ovarian cancer cell models respectively, revealed intracellular platinum concentrations of 12 fg/cell and 4 fg/cell, respectively which were 4-fold higher with respect to the uptake of cisplatin in both models. Intracellular drug release from the nanoconjugate was proved by measuring DNA platination in the same cells. In this case, levels of about 250 ng Pt/mg DNA were observed, about 5-fold higher when the nanoconjugate was used in comparison to cisplatin. Furthermore, the differences between the two lines turned to be significantly smaller than in the case of using cisplatin. The quantitative analytical tools developed here provided essential information required to fully characterize the developed nanoplatforms particularly important to overcome drug resistance.

Addressing the presence of biogenic selenium nanoparticles in yeast cells: analytical strategies based on ICP-TQ-MS.

Several organisms have demonstrated the ability of synthesising biogenic selenium-containing nanoparticles. Such particles from biological sources have attracted great attention due to several proven activities as antioxidants or antimicrobial agents. However, little is known in terms of size (distribution), shapes, chemical composition and number/amount/concentration of these particles. Therefore, in this work, we proposed the use of complementary analytical strategies that enabled the detection and characterization of selenium-containing nanoparticles in selenized yeast (Saccharomyces cerevisiae). The first strategy to address the intracellular presence of Se within yeast cells, involves the use of single cell ICP-TQ-MS (inductively coupled plasma-mass spectrometry). For this aim, selenium and phosphorous (as constitutive element) were measured as oxides (80Se16O+ and 31P16O+, resp.) in the triple-quadrupole mode. Then, a simple and fast cell lysis by mechanical disruption is conducted (approx. 30 min) in order to prove the presence of selenium-containing nanoparticles (SeNPs). The lysate is analysed by single particle ICP-TQ-MS and, complementarily, by liquid chromatography coupled to ICP-TQ-MS to cover a wider range of particle sizes. One of the samples revealed the presence of dispersed SeNPs with sizes between a few nm and up to 250 nm also confirmed by transmission electron microscopy (TEM) in the form of elemental selenium. The analysis of the certified reference material SELM-1 showed the presence of spherical SeNPs of 4 to 7 nm diameter. These biogenic particles, at least partially, were made of elemental selenium as well. The whole study reveals the excellent capabilities of "single" event ICP-MS methodologies in combination with HPLC-based strategies for a complete characterization of nanoparticulated material in biological samples.

Quantitative assessment of the metabolic products of iron oxide nanoparticles to be used as iron supplements in cell cultures.

Iron nanoparticles (NPs) metabolism is directly associated to human health due to their use as anemia treatment and should be studied in detail in cells. Here we present a speciation strategy for the determination of the metabolic products of iron oxide nanoparticles coated by tartaric and adipic acids in enterocytes-like cell models (Caco-2 and HT-29). Such methodology is based on the use of SDS-modified reversed phase high performance liquid chromatography (HPLC) separation using inductively coupled plasma-mass spectrometry (ICP-MS) as Fe selective detector. Post-column isotope dilution analysis is used as quantification tool by adding 57Fe as isotopically enriched standard. To assess the separation capability of the method, two different iron nanostructures: iron sucrose nanoparticles -Venofer®- used as model suspension and iron tartrate/adipate-modified nanoparticles, both of about 4 nm (core size) were evaluated. The two nanostructures were injected into the system showing good peak profiles and quantitative elution recoveries (>80%) in both cases. In addition, both nanoparticulate fractions could be based-line separated from ionic iron species, which needed to be complexed with 1 mM citrate to elute from the column. Exposed cells up to 0.5 mM of iron tartrate/adipate-modified nanoparticles were specifically treated to extract the internalized NPs and the extracts examined using the proposed strategy. The obtained results revealed the presence of three different fractions corresponding to nanoparticle aggregates, dispersed nanoparticles and soluble iron respectively in a single chromatographic run. Quantitative experiments (column recoveries ranging from 60 to 80%) revealed the presence of the majority of the Fe in the nanoparticulated form (>75%) by summing up the dispersed and aggregate particles. Such experiments point out the high uptake and low solubilization rate of the tartrate/adipate NPs making these structures highly suitable as Fe supplements in oral anemia treatments.

Single particle analysis of TiO2 in candy products using triple quadrupole ICP-MS.

Titanium dioxide (TiO2) belongs to the materials that have gained great importance in many applications. In its particulate form (micro- or nanoparticles), it has entered a huge number of consumer products and food-grade TiO2, better known as E171 within the European Union, represents an important food additive. Thus, there is an increasing need for analytical methods able to detect and quantify such particles. In this regard, inductively coupled-mass spectrometry (ICP-MS), in particular single particle ICP-MS (spICP-MS), has gained importance due to its simplicity and ease of use. Nevertheless, the number of applications for Ti nanoparticles is rather limited. In this study, we have applied the spICP-MS strategy by comparing different measuring modes available in triple quadrupole ICP-MS. First, single quadrupole mode using the collision/reaction cell system was selected for monitoring the isotope 47Ti. Different cell gases like He, O2 and NH3 were tested under optimised conditions for its applicability in spICP-MS of standard suspensions of TiO2. The determined analytical figures of merit were compared to those obtained by triple quadrupole mode using the 47Ti or 48Ti reaction products using O2 and NH3 as reaction gases. This comparison demonstrated that the triple quadrupole mode (TQ mode) was superior in terms of sensitivity due to the more efficient removal of spectral interferences. Particle size detection limits down to 26nm were obtained using the best instrumental conditions for TiO2 particles at a dwell time of 10ms. Finally, the different measuring modes were applied to the analysis of chewing gum samples after a simple extraction procedure using an ultrasonic bath. The obtained results showed a good agreement for the detected particle size range using the different TQ modes. The size range of TiO2 particles was determined to be between approximately 30 and 200nm, whereas roughly 40% of the particles were smaller than 100nm. For the determination of the particle number concentration in these real samples, we suggest CeO2 particles as internal standard.

Quantitative Evaluation of Cisplatin Uptake in Sensitive and Resistant Individual Cells by Single-Cell ICP-MS (SC-ICP-MS).

One of the main limitations to the Pt-therapy in cancer is the development of associated drug resistance that can be associated with a significant reduction of the intracellular platinum concentration. Thus, intracellular Pt concentration could be considered as a biomarker of cisplatin resistance. In this work, an alternative method to address intracellular Pt concentration in individual cells is explored to permit the evaluation of different cell models and alternative therapies in a relatively fast way. For this aim, total Pt analysis in single cells has been implemented using a total consumption nebulizer coupled to inductively coupled plasma mass spectrometric detection (ICP-MS). The efficiency of the proposed device has been evaluated in combination with flow cytometry and turned out to be around 25% (cells entering the ICP-MS from the cells in suspension). Quantitative uptake studies of a nontoxic Tb-containing compound by individual cells were conducted and the results compared to those obtained by bulk analysis of the same cells. Both sets of data were statistically comparable. Thus, final application of the developed methodology to the comparative uptake of Pt-species in cisplatin resistant and sensitive cell lines (A2780cis and A2780) was conducted. The results obtained revealed the potential of this analytical strategy to differentiate between different cell lines of different sensitivity to the drug which might be of high medical interest.

Enhanced detection of DNA sequences using end-point PCR amplification and online gel electrophoresis (GE)-ICP-MS: determination of gene copy number variations.

The design and evaluation of analytical methods that permit quantitative analysis of specific DNA sequences is exponentially increasing. For this purpose, highly sensitive methodologies usually based on labeling protocols with fluorescent dyes or nanoparticles are often explored. Here, the possibility of label-free signal amplification using end-point polymerase chain reaction (PCR) are exploited using on-column agarose gel electrophoresis as separation and inductively coupled plasma-mass spectrometry (ICP-MS) for the detection of phosphorus in amplified DNA sequences. The calibration of the separation system with a DNA ladder permits direct estimation of the size of the amplified gene fragment after PCR. With this knowledge, and considering the compound-independent quantification capabilities exhibited by ICP-MS for phosphorus (it is only dependent on the number of P atoms per molecule), the correlation of the P-peak area of the amplified gene fragment, with respect to the gene copy numbers (in the starting DNA), is then established. Such a relationship would permit the determination of copy number variations (CNVs) in genomic DNA using ICP-MS measurements. The method detection limit, in terms of the required amount of starting DNA, is ∼6 ng (or 1000 cells if 100% extraction efficiency is expected). The suitability of the proposed label-free amplification strategy is applied to CNVs monitoring in cells exposed to a chemical agent capable of deletion induction, such as cisplatin.

Evaluation of the biological effect of Ti generated debris from metal implants: ions and nanoparticles.

Metallic implants placed in humans exhibit wear and corrosion that result in the liberation of metal-containing by-products. In the case of titanium (Ti) containing implants, the metal containing debris may exist in a number of states, including metallic particles produced by mechanical wear and the products of metal corrosion in biological environments, such as the joints and surrounding fluids and tissues. In addition, these constituents may dissolve in both intracellular and extracellular solutions generating Ti ions. Both species, ions and nanoparticles, show different cellular toxicities. In this work we have evaluated the possible evolution of TiO2 nanoparticles (NPs) into soluble Ti metal ions by contact with biological fluids. For this aim, an in vitro study to address quantitative Ti solubilisation from TiO2 nanoparticles (with a diameter of 21 nm) after incubation with human serum at different concentrations has been conducted. Total Ti determination revealed low solubilisation rates ranging from 0.53 to 0.82% after just one week of incubation in the serum. The incubated serum was then subjected to speciation analysis by anion exchange liquid chromatography using an inductively coupled plasma mass spectrometer (ICP-MS) as an elemental detector for Ti monitoring. The obtained results revealed a significant increase in the Ti signal associated with the fraction of the protein transferrin and preferentially with one of the metal binding sites of the protein, the N-lobe. Thus, the effect of Ti at the cellular level has been evaluated by considering that it can be present either as ions or as nanoparticles using two different cells lines: human enterocytes HT29 and murine osteoblasts MC3T3. Significant toxicity was found at the highest concentration assayed (50 µg mL(-1)) for both Ti species (ions and NPs) and slightly higher for the ionic species at lower concentrations (1 and 10 µg mL(-1)).

Rapid separation of elemental species by multicapillary GC.

Multicapillary gas chromatography has been applied to the speciation of organomercury compounds. Basic investigation of the fundamental properties of multicapillary columns, to evaluate their potential and limitations as a rapid separation unit, are presented. For analysis of methylated and ethylated mercury compounds a complete separation can be performed within 45 s under isothermal conditions. The adaptation of the technique for use with a purge and trap system and with an element-selective plasma-emission detector results in a compact and effective system for mercury speciation. Results from analysis of certified reference materials were in good agreement with certified values within the significance levels.

Removal of interfering elements in ICP-QMS for the determination of Pt, Rh, and Pd by chemically modified sample introduction capillaries.

New on-line methods developed for the determination of Pt, Rh, and Pd by inductively coupled plasma-quadrupole mass spectrometry (ICP-QMS) include separation of elements which might lead to spectral interference in the quadrupole instrument. The fused-silica capillaries generally used for transport of the sample to mu-flow nebulizers have been chemically modified with ion-exchanger compounds to remove interfering elements such as Cu, Pb, or Hf. Characterization of the modification procedures by atomic-force microscopy showed that the quality of the quartz material and the kind of modification had a decisive influence on the yield of surface modification, and thus the exchange capacity of the capillaries.

Modification of capillaries coupled to micro-flow nebulizers: a new strategy for on-line interference removal in inductively coupled plasma mass spectrometry.

The application of interference removal within modified silica capillaries for the introduction of liquid samples by a micro-flow nebulizer into an inductively coupled plasma quadrupole mass spectrometer is described using the example of (103)Rh and its mass interferences resulting from (63)Cu (ArCu(+)), (87)Rb (RbO(+)), (87)Sr (SrO(+)) and (206)Pb (Pb(2+)). A strong cation exchanger was covalently bound to the capillary surface and its ability to interact with cations in aqueous solution was investigated. At pH 7. 0 the interfering elements can be selectively adsorbed within the capillaries without hampering the elution of Rh. The capillaries are easily regenerated by flushing with 10(-3) M hydrochloric acid in order to exchange retained cations for protons. Calibrations show no significant difference between the absence and presence of interfering elements, so the detection limit for Rh of 6.0 ng l(-1) (3sigma) is not influenced by adding up to a 100-fold excess of interferents. The method developed is compared with a mathematical correction model for inductively coupled plasma mass spectrometry using the example of Rh determination in particulate car exhaust fumes and the results obtained are discussed.

Validation of the determination of copper and zinc in blood plasma and urine by ICP MS with cross-flow and direct injection nebulization.

The simultaneous determination of copper and zinc in human plasma and urine by inductively coupled plasma mass spectrometry (ICP MS) is discussed. The performances of a cross-flow nebulizer and a direct-injection nebulizer (DIN) were compared. Flow-injection-DIN-ICP MS analysis of clinical samples using 1-2 mul samples was optimized. Isobaric interferences were discussed and were demonstrated to be eliminated for the (65)Cu and most of the Zn nuclides. The need for standard addition to compensate for signal suppression in the case of some serum samples was indicated. Results obtained by ICP MS using calibration with aqueous standard solutions were found to be in good agreement with those obtained by flame AAS for a batch of real blood plasma and urine samples. The methods developed were validated by analysis of several standard reference materials.

Speciation of mercury, platinum and tin - focus of research and future developments.

A discussion about the elements mercury, platinum and tin and their organometallic compounds, with special attention to important aspects like anthropogenic emissions, use in industry, agriculture and medicine, toxicities, biogeochemical cycles, highlights the necessity of speciation analysis. Methods frequently used to speciate mercury, platinum and tin compounds in different matrices are summarized. Future trends are indicated in order to give an impression about the importance of metal speciation and its increasing impact on some key disciplines.